Biliverdin regulates NR2E3 and zebrafish retinal photoreceptor development

NR2E3 is an orphan nuclear receptor whose loss-of-function causes abnormal retinal photoreceptor development and degeneration. However, despite that many nuclear receptors are regulated by binding of small molecule ligands, biological small molecule ligands regulating NR2E3 have not been identified. Identification of an endogenous NR2E3 ligand might reveal a previously unrecognized component contributing to retinal development and maintenance. Here we report that biliverdin, a conserved green pigment from heme catabolism, regulates NR2E3 and is necessary for zebrafish retinal photoreceptor development. Biliverdin from retinal extracts specifically bound to NR2E3’s ligand-binding domain and induced NR2E3-dependent reporter gene expression. Inhibition of biliverdin synthesis decreased photoreceptor cell populations in zebrafish larvae, and this phenotype was alleviated by exogenously supplied biliverdin. Thus, biliverdin is an endogenous small molecule ligand for NR2E3 and a component necessary for the proper development of photoreceptor cells. This result suggests a possible role of heme metabolism in the regulation of retinal photoreceptor cell development.

NR2E3, also known as photoreceptor-specific nuclear receptor (NR) or PNR, is a transcription factor enriched in developing and adult retina 1 . Mutations in the NR2E3 gene were found to cause inherited human diseases like Enhanced S-Cone Syndrome (ESCS) and retinitis pigmentosa 37 (RP37) [1][2][3] . These diseases are characterized by fewer rod photoreceptor cells with an increased number of short wavelength sensitive cone-like cells, suggesting a role of NR2E3 in retinal photoreceptor cell development and maintenance. Indeed, NR2E3, together with other retinal transcription factors like Crx, Nrl, and NR1D1, contributes to the regulation of photoreceptor-specific gene expression [4][5][6] . These functions of NR2E3 were also observed in other vertebrates like frogs and zebrafish 7,8 .
NR2E3 has also been suggested as a potential target to treat retinal disorders 9,10 . Forced expression of NR2E3 was shown to suppress the progression of retinitis pigmentosa in several mouse models 11 , while a synthetic NR2E3 antagonist suppressed the progression of retinitis pigmentosa caused by mutated rhodopsin 12 .
NR2E3 has a domain architecture shared by most other NRs 13 ( Figure S1A). It has an amino-terminal unstructured region harboring a trans-activator motif (AF1), followed by zinc-finger DNA-binding domain (DBD), a hinge region, and a carboxy-terminal ligand-binding domain (LBD) that harbors the second transactivation helix AF2. In the case of better characterized NRs, binding of small molecule ligand to LBD is a major regulatory mechanism 13 . Considering that a synthetic NR2E3 antagonist regulates NR2E3-dependent gene expression in cells and in animals 12 , we suspected that there might be a retinal small molecule regulating NR2E3 in vivo. At this moment, an endogenous NR2E3 ligand has not been identified, and NR2E3 remains as an orphan NR. Identification of an endogenous NR2E3 ligand might reveal a novel component contributing to retinal development and maintenance.
Here we report that biliverdin is an endogenous ligand regulating NR2E3 and is a compound that contributes to the development of zebrafish retina. Biliverdin is a conserved green pigment synthesized from heme by heme oxygenase (HO) isozymes 14 . HO isozymes are expressed in retina and other tissues, and their expressions are induced by light exposure 15 . Biliverdin is reduced to bilirubin by biliverdin reductase (BVR) isozymes 16 . We found that biliverdin from retinal extract specifically bound to NR2E3's ligand-binding domain (NR2E3 LBD ) in vitro. We also found that biliverdin induced NR2E3-dependent reporter gene expression in cells. Pharmacological inhibition of biliverdin synthesis in zebrafish larvae decreased the population of photoreceptor cells,

Identification of biliverdin's interaction with NR2E3 LBD in vitro. To gain insight into what might
regulate NR2E3 in retina, we searched for a retinal small molecule specifically binding to NR2E3 LBD . For this purpose, we used a combination of gel filtration chromatography and mass spectrometry 19,20 . Recombinant maltose-binding domain (MBP)-NR2E3 LBD (amino acid residues 164-410 of 410 residue NR2E3) fusion protein was expressed and purified from E. coli as in literature 21 . As previously reported 21 , without the MBP-tag, purified NR2E3 LBD was prone to aggregation. We thus used MBP-NR2E3 LBD without removing the tag. Retinal small molecules were extracted from rabbit retina using methanol. Macromolecules in the retinal extract were removed by passing the extract through a dialysis membrane (Fig. 1A). This pool of retinal small molecules was incubated with recombinant MBP-NR2E3 LBD . As control experiments, retinal small molecules were incubated without any protein or with recombinant MBP-NR2E1 LBD . NR2E1, also known as TLX, is an orphan NR closely related to NR2E3 22,23 . Retinal small molecules bound to MBP-NR2E3 LBD were separated from unbound small molecules by a gel filtration chromatography. Retinal small molecules co-purified with MBP-NR2E3 LBD were identified by a reverse-phase HPLC-mass spectrometry (RP-HPLC-MS  (Fig. 1B). These peaks were not observed when MBP-NR2E1 LBD was used instead of MBP-NR2E3 LBD (Fig. 1B). Commercial biliverdin showed a nearly identical RP-HPLC elution time and mass spectra ( Fig. 1B and Figure S1 in the Supplementary Information available with the online version of this paper). This result suggests that biliverdin (Fig. 1C) might be a retinal small molecule specifically binding to NR2E3 LBD .
Next, we measured the binding of biliverdin to MBP-NR2E3 LBD in vitro (Fig. 2). Because biliverdin is a colored compound, the binding of biliverdin to MBP-NR2E3 LBD was measured by monitoring the visible spectrum of biliverdin while varying the concentration of MBP-NR2E3 LBD ( Fig. 2A). Free biliverdin has two distinct absorption peaks-one in the blue region and the other in the red region. In the presence of MBP-NR2E3 LBD , the blue absorption peak was shifted to a longer wavelength (from 390 to 430 nm) while the red peak intensity was decreased ( Fig. 2A). This spectral change of biliverdin induced by MBP-NR2E3 LBD is distinct from the spectra of biliverdin bound to albumin 24 or phytochrome proteins 25 . When small molecules from the biliverdin-MBP-NR2E3 LBD complex were recovered and analyzed by RP-HPLC-MS, the HPLC retention time and the massto-charge ratio of the recovered compound was identical to free biliverdin ( Figure S2). This result indicates that the spectral change of biliverdin bound to MBP-NR2E3 LBD is reversible upon the removal of the protein. Biliverdin and NR2E3 LBD formed a stoichiometric complex. A plot of the blue absorbance peak intensity against www.nature.com/scientificreports/ the MBP-NR2E3 LBD concentration showed that biliverdin stoichiometrically bound to MBP-NR2E3 LBD with a K D of 0.2 μM (Fig. 2B). Next, we asked whether an Enhanced S-Cone Syndrome (ESCS) mutation affects biliverdin's binding to NR2E3 LBD . A blind-docking simulation of the biliverdin-NR2E3 LBD interaction suggested that the compound bound to a pocket adjacent to the R311 residue ( Figure S3). We chose the R311Q mutation because this mutation is also a pathological mutation frequently found in ESCS patients (ClinVar accession number VCV000005532). We found that MBP-NR2E3 LBD R311Q variant was poorly bound to biliverdin (Fig. 2B, calculated K D 9 μM). This result indicates that the R311 residue is necessary for biliverdin to bind to NR2E3 LBD , and suggests that the interaction of biliverdin and NR2E3 might be relevant to the ESCS phenotype.
This interaction of biliverdin and MBP-NR2E3 LBD was specific. First, biliverdin did not show any evidence of binding to MBP-NR2E1 LBD at micromolar concentrations (Fig. 2B). The NR2E1 LBD protein sequence is ~ 48% identical to NR2E3 LBD , and it is a closest neighbor of NR2E3 among members of the NR family proteins ( Figure  S3). Second, NR2E3 LBD distinguished biliverdin from similar compounds (Fig. 2C). The visible spectrum of bilirubin was little affected by MBP-NR2E3 LBD until the protein concentration reached nearly 10 μM. Unlike peroxisome proliferator-activated reporter alpha (PPARalpha), which is an NR binding to both bilirubin and biliverdin at micromolar concentrations 24 , NR2E3 is more specific to biliverdin. Considering that serum concentration of bilirubin is higher than that of biliverdin, this selectivity of NR2E3 toward biliverdin over bilirubin might be physiologically relevant. We also observed that protoporphyrin did not show any evidence of binding to MBP-NR2E3 LBD at micromolar concentrations (Fig. 2C). Taken together, these results indicated that the biliverdin is a specific ligand for NR2E3 LBD at least in vitro.
This interaction between biliverdin and NR2E3 might be conserved in other organisms. Zebrafish NR2E3 LBD is ~ 73% identical to human NR2E3 LBD at the amino acid sequence level ( Figure S4). When the visible spectrum www.nature.com/scientificreports/ of biliverdin was measured varying the concentration of zebrafish NR2E3 LBD (MBP-DrNR2E3 LBD ), the spectrum of biliverdin showed changes similar to the one observed with human NR2E3 LBD (Fig. 2D). This result suggests that the binding of biliverdin may be conserved in other organisms.
Biliverdin induced NR2E3-dependent reporter gene expression. Next, we asked whether biliverdin affects transcriptional activity of NR2E3 in cells. For this purpose, we used a reporter gene expression in 293F cells. Because serum can contain variable amounts of biliverdin (0.9-6.5 µM total biliverdin, that includes free biliverdin and ones bound to blood proteins like albumin 26 ), we used serum-free chemically defined medium in this experiment. We found that biliverdin (0.1 μM) significantly (p < 0.01) induced the NR2E3-dependent reporter gene expression by ~ threefold (Fig. 3A). At the tested concentration (0.1 μM), biliverdin only marginally affected the NR2E3 R311Q-dependent reporter gene expression, which reflects our in vitro results (Fig. 3A). When the experiment was repeated varying the concentration of biliverdin, the effective concentration at 50% of the maximum effect (EC 50 ) was calculated to be 5 nM (Fig. 3B). At concentrations above 10 μM, biliverdin affected the viability of 293 cells. We thus carried out subsequent cellular and in vivo experiments at lower concentrations of biliverdin. These results indicated that biliverdin regulates NR2E3's function in cells and that the loss of this interaction might be a reason behind the ESCS phenotype observed with the R311Q mutation. Next, we asked whether biliverdin affects NR2E3 in cells naturally expressing NR2E3. We thus tested effects of biliverdin on WERI-Rb-1 27 and Y79 retinoblastoma cells. Although retinoblastoma cells do not recapitulate all aspects of retinal photoreceptor cells, NR2E3 is expressed in retinoblastoma cells 28 and regulates its own expression 29 . Our Western blot analysis of NR2E3 in retinoblastoma cells showed that biliverdin induced the level of NR2E3 protein by approximately twofold (2.0 ± 0.2 fold elevation by 1 μM biliverdin, n = 3; Fig. 3C, S5) in a dose-dependent manner (Fig. 3D, not repeated). Taken together with the reporter gene expression analysis, this result suggests that biliverdin regulates NR2E3 levels in cells. www.nature.com/scientificreports/ Biliverdin regulates photoreceptor development in zebrafish larvae. Because of the importance of NR2E3 in retinal photoreceptor cell development, we tested whether biliverdin is required for retinal photoreceptor cell development. Several reasons made zebrafish a good model system for this purpose [30][31][32] . First, zebrafish eyes are similar to humans 30 . Second, the necessity of NR2E3 in zebrafish photoreceptors has also been demonstrated 7 . Third, monitoring early retinal developmental processes is feasible with zebrafish embryos. Finally, delivery of small molecules to retina can be more readily achieved in zebrafish embryos than mammals 31 . Because there are multiple isoforms of HO isozymes and BVR isozymes in zebrafish, we used a pharmacological HO inhibitor to suppress biliverdin synthesis. Sulconazole nitrate (SN) inhibits HO isozymes in vitro and in vivo and would be suitable for this purpose 33 . At tested concentrations, neither SN nor exogenously supplied biliverdin caused observable developmental defects in zebrafish embryos (Fig. 4A, B). In contrast, fluorescence microscopic analysis of retinas from XOPS-GFP transgenic larvae (which express GFP specifically in rod photoreceptors) showed an approximate 30% decrease in the number of rod photoreceptor cells in SN treated larvae at 3 days post fertilization (dpf; p = 0.0172, untreated n = 7, SN-treated n = 13; Fig. 4C, D). This decreased rod cell population was also reported to occur in nr2e3-ko zebrafish larvae 7 . This reduced rod phenotype in SN-treated zebrafish larvae was alleviated by exogenously supplied biliverdin (Fig. 4C, D). This result indicates that the decreased rod cell population was not because of the accumulation of heme but because of loss of biliverdin or its downstream product.
Interestingly, the pharmacological inhibition of biliverdin synthesis showed broader impacts on photoreceptor cell development than were reported for the zebrafish nr2e3-ko. We found that the population of red-green cone photoreceptors (immunolabeled with the Zpr-1 antibody), was also decreased by sulconazole nitrate at 3 dpf (p < 0.001; for rods: untreated n = 5, sulconazole nitrate-treated n = 6; for cones: untreated n = 5, sulconazole nitrate-treated n = 11; Fig. 4E, F). Exogenously provided biliverdin increased the cone photoreceptor cell population (Fig. 4E, F), demonstrating that this phenotype was specific to the loss of biliverdin. A similar decrease of immature rod photoreceptors (immunolabeled with the 4C12 antibody) was also observed following SN exposure (Fig. 4E, F). Taken together, these results suggest that, in addition to the regulation of NR2E3 in rod precursors, biliverdin might be required for photoreceptor specification or differentiation upstream to the differentiation of rod photoreceptor cells, perhaps in pan-photoreceptor progenitors.

Discussion
NR2E3 has been implicated in the development and the maintenance of retinal photoreceptor cells. Identifying its in vivo ligand is important in two aspects. First, the identity of an NR2E3 ligand provides a novel component contributing to retinal development. Second, this ligand can serve as a template to develop a reagent for translational therapeutic purposes.
Our results provide another piece of data connecting heme metabolism to retinal development. In addition to NR2E3, there are several NRs involved in retinal development 34 . One NR playing an important role in retinal photoreceptor cell development and beyond is NR1D1 (also known as Rev-Erb alpha), which functions in concert with NR2E3 in retinal development while having an additional role in circadian rhythm. Heme itself is a ligand regulating NR1D1 and circadian rhythm 17,35,36 . In this study we show that biliverdin, the immediate downstream product of heme, regulates NR2E3, further suggesting a connection between heme metabolism and retinal photoreceptor transcription network. Our results suggest that inhibition of biliverdin synthesis results in a broader impact on zebrafish photoreceptor development than what was reported with nr2e3-ko animals; both rod and cone photoreceptors were reduced at 3 dpf in biliverdin-inhibited larvae, whereas only rods were affected at early stages in nr2e3-ko zebrafish, although the cones eventually showed signs of degeneration in adults 7 . Further analysis at later differential stage might be informative. We speculate that this expanded role for biliverdin in photoreceptor development may be due to additional binding to other NR proteins by biliverdin or its downstream metabolites in photoreceptor progenitors. Interestingly, no increase in short-wavelength cones was observed in the nr2e3-ko zebrafish study (in contrast to the mouse and human photoreceptor phenotypes resulting from loss of Nr2e3); since our zebrafish experiments only analyzed the numbers of red and green cones, it remains to be determined whether loss of biliverdin could also result in an increase in short-wavelength (blue and UV) cones in zebrafish.
Why biliverdin? We suspect that there might be a connection to light and/or oxidative stress response. As a light-absorbing molecule, biliverdin is used as a chromophore sensing red light in plants and some bacteria 25,37 . Our spectroscopic result, however, suggests that biliverdin-NR2E3 has a spectral property distinct from free biliverdin or plant phytochromes. This spectral property of biliverdin-NR2E3 makes it unlikely to sense red light as in plant phytochromes. Instead, it is possible that biliverdin-NR2E3 may sense blue light. In addition, biliverdin can function as a lipophilic redox signaling molecule. Excess heme can cause oxidative damage to light-exposed cells, and HO isozymes protect these cells from oxidative stress [38][39][40][41] . In general, this protective function of HO isozymes is believed due to the removal of excess hemes. By binding to biliverdin, the product of HO isozymes, NR2E3 may indirectly sense light and oxidative stress. Indeed, in non-retinal MCF-7 breast cancer cells, oxidative stress-causing benzopyrene reduced the expression of NR2E3, which is rescued by antioxidant 42 . In MCF-7 cells, NR2E3 regulates the expression of aryl hydrocarbon receptor, implying relevance to NR2E3 in oxidative stress response in these cells 43 . At this stage, it is not clear whether biliverdin-NR2E3 directly or indirectly senses light.
NR2E3 is expressed in the adult as well as the developing retina. Genetically overexpressing NR2E3 showed beneficial effects in several different retinitis pigmentosa models, suggesting that an increased NR2E3 activity can be beneficial 11 . We speculate that biliverdin or its derivatives might be useful in the development of reagents targeting retinitis pigmentosa. www.nature.com/scientificreports/  Table S1 available with the online version of this paper. Plasmids constructed for this study were from GenScript and sequenced from both ends of inserts. Proteins were expressed and purified from BL21 (DE3) CodonPlus RILP cells (Agilent) harboring appropriate plasmids. For the purification of MBP-NR2E3 LBD , cells were grown in 1-L Terrific Broth supplemented with 50 µg/mL ampicillin and 34 µg/mL chloramphenicol at 37 °C until OD 600 became 1. Protein expression was induced by the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to 0.5 mM, followed by an overnight incubation at 12 °C. Cells were harvested by centrifugation (5000 g for 20 min at 4 °C), and the pellet was resuspended in 20 mL buffer 1 (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride or TCEP, 10 mM imidazole, 0.01% IGEPAL CA-630) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by sonication and cleared by centrifugation (15,000 g for 20 min at 4 °C). The supernatant was loaded onto a 5 mL His-Trap column (Cytivia) pre-equilibrated in buffer 1. The column was washed with 50 mL buffer 1, and bound proteins were eluted with buffer 1 containing 250 mM imidazole. The eluted proteins were concentrated using Amicon Ultracel-15 (molecular weight cut off 10 kDa) and dialyzed several times against 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.01% IGEPAL CA-630. MBP-NR2E1 LBD was also prepared using an identical method.  Zebrafish treatment, cryosections, immunohistochemistry and cell counts. Embryos were generated from XOPS:GFP in-crosses and randomly subdivided into groups of 5 at 48 h post fertilization (hpf). Each group was placed in one of 5 treatments in fish water: untreated, 0.1% DMSO, 0.5 µM sulconazole nitrate, 0.5 µM biliverdin, and 0.5 µM sulconazole nitrate + 0.5 µM biliverdin. At 72 hpf, embryos were fixed in 4% paraformaldehyde overnight, then incubated in 10% followed by 30% sucrose at 4 °C. Sectioning and immunohistochemistry were conducted as previously described 46 and immunolabeled sections were imaged on either a Nikon inverted (Nikon Ti-U) or confocal microscope (Leica SP8, Leica). The following antibodies were used: anti-4C12 (immature and mature rod photoreceptors, mouse, 1:100, provided by James Fadool, Florida State University) and anti-Zpr1 (red/green cone photoreceptors, 1:20, mouse, ZIRC). Slides were incubated in 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei (1:10,000 dilution, Sigma). Photoreceptor cells were quantified by counting individual cells and at least 5 embryos were used for each analysis, across 3 biological replicates. Statistics were conducted using an one-way ANOVA followed by post-hoc Tukey test using GraphPad software. P-values less than 0.05 were considered significant and are indicated by *, p < 0.01 is indicated by **, and p < 0.001 by ***. Boxplots were generated using R (version 3.6.2), R studio (version 1. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.